Adrenocortical carcinoma (ACC) is very rare with an incidence of 1 per million. This contrasts with the much more common benign adrenal tumors (adenomas) which are incidentally found in up to 3-10% of patients undergoing radiologic imaging (CT or MRI). However, unlike their common benign counterparts, ACC has a dismal prognosis with an overall 5-year survival of only 15-30 %. While incidentally discovered adrenal adenomas rarely progress to ACC, this concern mandates one or more repeated imaging studies with additional patient radiation exposure and cost. Furthermore, there are no biomarkers that have proven useful for early detection, monitoring progression or post- surgical reoccurrence of ACC. We propose to define biomarkers that will differentiate ACC from benign adrenal tumors through the research proposed in three specific aims. Specific Aim 1 will define the ACC steroid- metabolizing enzyme transcriptome. Specific Aim 2 will define the ACC steroid metabolome. Specific Aim 3 will develop a set of parameters based on steroid profiles that reliably differentiates ACC patients from those with benign adrenal tumors. Impact: This application directly addresses the need for clinically applicable biomarkers for ACC. Upon completion of this project, we expect to have assays that can be validated in larger cohort clinical trials. Currently, no biomarkers reliably differentiate benign from malignant adrenal tumors. Hence, there is a critical need to identify an ACC steroid signature that could be used for patient screening, as well as monitoring for ACC progression or tumor recurrence. ACC biomarkers will simplify screening and improve patient safety for the thousands of patients found to have adrenal nodules, while also enhancing the care of ACC patients.